Cancer is an aberrant net accumulation of atypical cells, which can result from an excess of proliferation, an insufficiency of cell death, or a combination of the two.
Proliferation is the culmination of a cell's progression through the cell cycle resulting in the division of one cell into two cells. The 5 major phases of the cell cycle are G0, G1, S, G2 and M. During the G0 phase, cells are quiescent. Most cells in the body, at any one time, are in this stage. During the G1 phase, cells, responding to signals to divide, produce the RNA and the proteins necessary for DNA synthesis. During the S-phase (SE, early S-phase; SM, middle S-phase; and SL, late S-phase) the cells replicate their DNA. During the G2 phase, proteins are elaborated in preparation for cell division. During the mitotic (M) phase, the cell divides into two daughter cells. Alterations in cell cycle progression occur in all cancers and may result from over-expression of genes, mutation of regulatory genes, or abrogation of DNA damage checkpoints (Hochhauser D. Anti-Cancer Chemotherapeutic Agents 8:903, 1997).
Unlike cancer cells, most normal cells cannot proliferate indefinitely due to a process termed cellular senescence. Cellular senescence is a programmed cell death response leading to growth arrest of cells (Dimri et al. Proc. Natl. Acad. Sci. USA 92:20, 1995). DNA damage, exposure of colon, breast and ovarian cancer cells to toposiomerase inhibitors and exposure of nasopharyngeal cancer cells to cisplatin are reported to prevent proliferation of these cells by induction of senescence (Wang et al. Cancer Res. 58:5019, 1998; Poele et al. Br. J. Cancer 80:9, 1999).
Synthetic oligonucleotides are polyanionic sequences that are internalized in cells (Vlassov et al. Biochim. Biophys. Acta 1197:95, 1994). Synthetic oligonucleotides are reported that bind selectively to nucleic acids (Wagner, R. Nature: 372:333, 1994), to specific cellular proteins (Bates et al. J. Biol. Chem. 274:26369, 1999) and to specific nuclear proteins Scaggiante et al. Eur. J. Biochem. 252:207, 1998) to inhibit proliferation of cancer cells.
Synthetic 27 base sequences containing guanine (G) and variable amounts of thymine (T) (oligonucleotide GTn), wherein n is ≧1 or ≦7 and wherein the number of bases is ≧20 (Scaggiante et al. Eur. J. Biochem. 252:207, 1998), are reported to inhibit growth of cancer cell lines by sequence specific binding to a 45 kDa nuclear protein, whereas GTn, wherein the number of bases is ≦20, are reported to be inactive against cancer cell lines (Morassutti et al. Nucleosides and Nucleotides 18:1711, 1999). Two synthetic GT-rich oligonucleotides of 15 and 29 bases with 3′ aminoalkyl modifications are reported to form G-quartets that bind to nucleolin and to inhibit proliferation of cancer cell lines (Bates et al. J. Biol. Chem. 274:26369, 1999). The synthetic 6 base TTAGGG-phosphorothioate, having a sequence identical to that of the mammalian telomere repeat sequence, is reported to inhibit proliferation of Burkitt's lymphoma cells in vitro and in vivo (Mata et al. Toxicol. Applied Pharmacol. 144:189, 1997). However, the synthetic 6 base TTAGGG-phosphodiester is reported to have no anti-telomerase activity (U.S. Pat. No: 5,643,890).
Cell death is effected by immune-mediators that promote apoptosis, and by apoptosis inducers that directly initiate pathways leading to cell death (Muzio et al. Cell 85:817, 1996; Levine, A. Cell 88:323, 1997). Apoptosis is an active cellular death process characterized by distinctive morphological changes that include condensation of nuclear chromatin, cell shrinkage, nuclear disintegration, plasma membrane blebbing, and the formation of membrane-bound apoptotic bodies (Wyllie et al. Int. Rev. Cytol. 68:251, 1980). A molecular hallmark of apoptosis is degradation of the cell's nuclear DNA into oligonucleosomal-length fragments as the result of activation of endogenous endonucleases (Wyllie A. Nature 284:555, 1980).
Caspases (cysteine-aspartyl-specific proteases) have been implicated as key enzymes in the execution of the late stage of apoptosis. The caspase family consists of at least fourteen related cysteine aspartyl proteases. All the caspases contain a conserved QACXG (where X is R, Q or G) pentapeptide activesite motif (Cohen G. Biochim. Biophys. Acta 1366:139, 1997). A number of caspases are synthesized as inactive proenzymes that are activated following cleavage at caspase specific cleavage sites (Cohen G. Biochim. Biophys. Acta 1366:139, 1997) or as inactive enzymes that require association with regulatory molecules for activation (Stennicke et al. J. Biol. Chem. 274:8359, 1999).
In addition to their role in apoptosis, caspases are involved in activation and proliferation of B and T lymphocytes, in cytokine maturation during inflammation, in differentiation of progenitor cells during erythropoiesis and in development of lens fiber (Fadeel et al. Leukemia 14:1514, 2000). With respect to B and T lymphocytes, caspase 3 is processed during activation of B lymphocytes and of CD4 (+), CD8 (+), CD45RA(+) and CD45RO (+) subsets of T lymphocytes (Alam et al. J. Exp. Med. 190:1879, 1999). Moreover, stimulation of T lymphocytes by mitogens and by interleukin-2 is associated with activation of the caspase pathway and with cleavage of PARP (Wilheim et al. Eur. J. Immunol. 28:891, 1998). With respect to cytokines, caspase 3 activity is necessary for the release of IL-2 by activated T lymphocytes (Posmantur et al. Exp. Cell Res. 244:302, 1998) and for the processing and maturation of the pro-inflammatory cytokine interleukin-16 (Zhang et al. J. Biol. Chem. 273:1144, 1998). With respect to erythropoiesis, caspase activation is involved in erythropoiesis regulation and has been shown to modulate GATA-1, a nuclear regulatory protein crucial for the maturation of erythroid precursors (De Maria, et al. Nature 401:489, 1999).
Cytolysis is the complete or partial destruction of a cell and is mediated by the immune system. Activated macrophages and monocytes produce bioactive molecules that include, but are not limited to cytokines. Cytokines, include, but are not limited to, interleukin (IL)-1, IL-1 beta, IL-6, IL-10, IL-12, and TNF-alpha.
IL-1 beta reduces bone marrow cell sensitivity to cytoreductive drugs, to radiation and to in vitro tumor cell purging with drugs in autologous bone marrow transplantation (Dalmau et al. Bone Marrow Transplant. 12:551, 1993).
IL-6 induces B cell differentiation, stimulates IgG secretion (Taga et al. J. Exp. Med. 166:967, 1987), induces cytotoxic T cell differentiation (Lee et al. Vaccine 17:490, 1999), promotes megakaryocyte maturation (Ishibashi et al. Proc. Natl. Acad. Sci. USA 86:8953, 1989) and functions both as an anti-proliferative factor (Mori et al. Biochem. Biophys. Res. Comm. 257:609, 1999; Alexandroff et al. Biochem. Soc. Trans. 25:270, 1997; Takizawa et al. Cancer Res. 53:18, 1993: Novick et al. Cytokine 4:6, 1992) and as a pro-proliferative factor (Okamoto et al. Cancer Res. 57:141, 1997; Okamoto et al. Int. J. Cancer 72:149, 1997; Chiu et al. Clin. Cancer Res. 2:215, 1996; Lu et al. Clin. Cancer Res. 2:1417, 1996) for cancer cells.
IL-10 enhances the effectiveness of vaccines in murine tumor models (Kauffman et al. J. Immunother. 22: 489, 1999) and up-regulates anti-cancer autoreactive T cell responses (Alleva et al. Immunobiol. 192:155, 1995).
IL-12, alone and in combination with other cytokines, promotes the maturation of leukocytes and induces the secretion of interferon-gamma. IL-12 is reported to have anti-cancer activity (Stine et al. Annals NY Academy of Science 795:420, 1996; Chen et al. Journal of Immunol. 159:351, 1997) including, but not limited to, activation of specific cytolytic T-lymphocytes, activation of natural killer (NK) cells and induction of the anti-angiogenic proteins IP-10 and MiG.
TNF-alpha causes necrosis of solid tumors (Porter et al. Trends in Biotech. 9:158, 1991), sensitizes cancer cells to gamma irradiation-induced apoptosis (Kimura et al. Cancer Res. 59:1606, 1999) and protects bone marrow precursor cells from the effects of antineoplastic agents (Dalmau et al. Bone Marrow Transplant. 12:551, 1993).
However, most prior art anti-cancer therapies, whether directed to induction of cell cycle arrest, inhibition of proliferation, induction of apoptosis or stimulation of the immune system, have proven to be less than adequate for clinical applications. Many of these therapies are inefficient or toxic, have significant adverse side effects, result in development of drug resistance or immunosensitization, and are debilitating for the recipient
Therefore, there is a continuing need for novel compositions and methods that induce cell cycle arrest in cancer cells, inhibit proliferation of cancer cells, activate caspases in cancer cells, induce apoptosis in cancer cells and stimulate cytokine production by immune system cells.